Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context­-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

21Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the 22 engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have 23 enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed 24 mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date 25 been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of 26 inducible transcriptional regulators in these strains also prevents concurrent control of genome 27 engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, 28BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity 29 and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination 30 efficiency, and (3) four independent inducible regulators to control engineered functions. These 31 modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold 32 increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To 33 facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci,34 termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is 35 a platform to develop and optimize engineered cellular functions and can serve as a model to implement 36 comparable recombination and regulatory systems in other bacteria. 37 INTRODUCTION 38The design-build-test (DBT) cycle is a common paradigm used in engineering disciplines. Within the 39 context of synthetic biology it is employed to engineer user-defined cellular functions for applications 40 such as metabolic engineering, biosensing, and therapeutics (1, 2). The rapid prototyping of engineered 41 functions has been facilitated by advances in in vitro DNA assembly, and plasmids have traditionally 42 been used to implement designs in vivo given their ease-of-assembly and portability. However, for 43 deployment in contexts beyond the laboratory such as large-scale industrial bioprocesses or among 44 complex microbial communities, plasmid-based circuits suffer from multiple limitations: high intercellular 45 variation in gene expression, genetic instability from random partitioning of plasmids during cell division, 46 and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or 47 is economically infeasible (3, 4). These shortcomings can be ameliorated once a design is transferred 48 from a plasmid to the host genome, which offers improved genetic stability and lower expression 49 variation (5) along with reduced metabolic load (6). However, behaviors optimized for plasmid contexts 50 often do not map predictably to the genome. As such, building and testing designs directly on the 51 genome can reduce the DBT cycle time and facilitate engineering cellular programs for complex 52 e...

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σB, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, σA, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from σB-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between σB- and σA-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.

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The genus Amaranthus is composed of numerous annual herbs, several of which are primary driver weeds within annual production agricultural systems. In particular, Amaranthus tuberculatus, a dioecious species, is noteworthy for rapid growth rates, high fecundity, and an expanding geographic distribution. Interspecific hybridization within and between the subgenera Amaranthus and Acnidia is reported both in controlled environment and field studies, however a gap in knowledge exists with the subgenus Albersia. Interspecific hybridization may contribute to genetic diversity, and may contribute to the current range expansion of A. tuberculatus. Recently, a herbicide resistance survey of A. tuberculatus across five Midwestern states reported alleles of PPX2 similar to sequences of Amaranthus albus, a monoecious species. Here, we seek to generate empirical data for the hybridization potential of A. albus and A. tuberculatus through replicated, controlled crosses in a greenhouse. Of 65,000 progeny screened from A. albus grown with A. tuberculatus males, three were confirmed as hybrids. Hybrids were dioecious, possessed phenotypic traits of both species, and had limited to no fertility. DNA content analysis of backcross progeny suggested a polyploid state may be required for hybrid formation. Screening of 120 progeny of A. tuberculatus females grown with A. albus identified no hybrids, though a skew to female progeny was observed. The female skew may be due to apomixis or auto-pollination, the spontaneous generation of male flowers on otherwise female plants. Our results indicate that introgression between A. albus and A. tuberculatus will occur less frequently than what has often been reported from hybridization studies with different pairs of Amaranthus species.